EGR1 as a potential marker of prognosis in extranodal NK/T-cell lymphoma.

Extranodal natural killer T-cell lymphoma (ENKTL) is an aggressive malignancy with a dismal prognosis. In the present study, gene expression profiling was performed to provide more information on ENKTL molecular signature and offer a rationale for further investigation of prognostic markers in ENKTL. NanoString nCounter Analysis encompassing 133 target genes was used to compare gene expression levels of 43 ENKTL tumor samples. The majority of the patients were under 60 years of age (79.1%); 32 (74.4%) patients had nasal type ENKTL and 23 patients (53.5%) had intermediate/high risk ENKTL based on the prognostic index for natural killer cell lymphoma (PINK). The median follow-up was 15.9 months and the median overall survival (OS) was 16.1 months (95% CI 13.0-69.8). EGR1 upregulation was consistently identified in the localized stage with a low risk of prognostic index based on the PINK. Among the six significantly relevant genes for EGR1 expression, high expression levels of genes, including CD59, GAS1, CXCR7, and RAMP3, were associated with a good survival prognosis. The in vitro test showed EGR1 modulated the transcriptional activity of the target genes including CD59, GAS1, CXCR7, and RAMP3. Downregulation of EGR1 and its target genes significantly inhibited apoptosis and decreased chemosensitivity and attenuated radiation-induced apoptosis. The findings showed EGR1 may be a candidate for prognostic markers in ENKTL. Considerable additional characterization may be necessary to fully understand EGR1.

Sex differences in c-Fos and EGR-1/Zif268 activity maps of rat sacral spinal cord following cystometry-induced micturition.

Storage and voiding of urine from the lower urinary tract (LUT) must be timed precisely to occur in appropriate behavioral contexts. A major part of the CNS circuit that coordinates this activity is found in the lumbosacral spinal cord. Immediate early gene (IEG) activity mapping has been widely used to investigate the lumbosacral LUT-related circuit, but most reports focus on the effects of noxious stimulation in anesthetized female rats. Here we use c-Fos and EGR-1 (Zif268) activity mapping of lumbosacral spinal cord to investigate cystometry-induced micturition in awake female and male rats. In females, after cystometry c-Fos neurons in spinal cord segments L5-S2 were concentrated in the sacral parasympathetic nucleus (SPN), dorsal horn laminae II-IV, and dorsal commissural nucleus (SDCom). Comparisons of cystometry and control groups in male and female revealed sex differences. Activity mapping suggested dorsal horn laminae II-IV was activated in females but showed net inhibition in males. However, inhibition in male rats was not detected by EGR-1 activity mapping, which showed low coexpression with c-Fos. A class of catecholamine neurons in SPN and SDCom neurons were also more strongly activated by micturition in females. In both sexes, most c-Fos neurons were identified as excitatory by their absence of Pax2 expression. In conclusion, IEG mapping in awake male and female rats has extended our understanding of the functional molecular anatomy of the LUT-related circuit in spinal cord. Using this approach, we have identified sex differences that were not detected by previous studies in anesthetized rats.

Short-term but not long-term exposure to an enriched environment facilitates the extinction of aversive memory.

Environmental enrichment (EE) has been used to investigate behavioral changes and neuroplasticity in brain in normal and pathological conditions. Besides, the EE has been used to understand the neurobehavioral systems involved in learning experiences, visual inputs, defensive responses, social interactions and memory. However, the required exposure duration to remove aversive memories remains lacking. Therefore, the purpose of the present study was to investigate the time-course effect of EE exposure on the extinction of aversive memory. Young adult male Wistar rats were exposed to two different EE protocols: short-term environmental enrichment (EE2 - animal kept under enriched conditions for two weeks) and long-term environmental enrichment (EE4 - animal kept under enriched conditions for four weeks). The contextual fear conditioning test was used to assess aversive memory. The both EE protocols provide changes in Zif-268 immunoreactivity in mesocorticolimbic areas such as CA1 and central amygdala; however, only short-term EE reduces the ZIF-268 immunoreactivity in VTA. Besides, both EE protocols also provide an increase in TH immunoreactivity in VTA and nucleus accumbens, but only the short-term EE modifies the TH immunoreactivity in CA1 and infralimbic region of the prefrontal cortex. The time-course effect of EE interferes differently on the extinction of aversive memory, being two weeks of exposure with EE sufficient to cause improvement in coping during aversive situations, favoring the extinction of conditioned fear memory.

Monitoring from Battlefield to Bedside: Serum Repositories Help Identify Biomarkers, Perspectives on Mild Traumatic Brain Injury.

OBJECTIVES: Serum repositories are foundations for seroepidemiological data, revealing targeted information about morbidities and existing heterogeneity in human populations. With the recent technological advances, we can perform high-throughput screening at an affordable cost using minimal plasma. Monitoring brain health after an injury is critical since mild Traumatic Brain Injury (mTBI) and other neurological symptoms are under-diagnosed. Our objective in this study is to present our preliminary serological data from one of our ongoing studies on mTBI. METHODS: In this retrospective study, we used stored plasma samples to understand biomarkers of mTBI. We compared plasma samples from five patients with mTBI following their first concussive episode to five gender and age-matched healthy controls. We assessed multiple biomarkers to show the importance of biorepositories. RESULTS: Most of the estimated plasma factors in mTBI subjects at baseline were comparable to normal healthy individuals except for the astroglial markers S100B and glial fibrillary acidic protein. Fluctuations of these biomarkers can affect the homeostasis of brain parenchyma by altering the neural network signaling, which in turn may result in intermittent behavioral symptoms. CONCLUSION: Biorepositories are powerful resources for understanding the spectrum of morbidity. Biomarkers serve as a valuable diagnostic and therapeutic tool.

Detection of zinc finger protein (EGR1) based on electrogenerated chemiluminescence from singlet oxygen produced in a nanoclay-supported porphyrin environment.

Early growth response protein 1 (EGR1), as a characteristic example of zinc finger proteins, acts as a transcription factor in eukaryotic cells, mediating protein-protein interactions. Here, a novel electrochemiluminescence (ECL)-based protocol for EGR1 assay was developed with a new eco-friendly emitter: singlet oxygen produced in the vicinity of nanoclay-supported zinc proto-porphyrin IX (ZnPPIX). Its electrochemical reduction stimulates an intense monochromic CL irradiation at 644 nm from the dissolved oxygen as endogenous coreactant in the aqueous solution. This ECL derivation was rationalized via hyphenated spectroscopy and theoretical calculation. To promote hydrophilicity and solid-state immobilization of porphyrins, the lamellar artificial laponite was employed as a nanocarrier owning to its large specific area without the blackbody effect. The facile exfoliation of laponite produced quality monolayered nanosheets and facilitated the adsorption and flattening of PPIX upon the surface, resulting in a highly efficient ECL emission. Based on the release of Zn(2+) in zinc finger domains of EGR1 upon contact with the ECL-inactive PPIX, which was monitored by circular dichroism and UV-absorption, a sensitive Zn(2+)-selective electrode for the "signal-on" detection of EGR1 was prepared with a detection limit down to 0.48 pg mL(-1) and a linearity over 6 orders of magnitude. The proposed porphyrin-based ECL system thus infused fresh blood into the traditional ECL family, showing great promise in bioassays of structural Zn(II) proteins and zinc finger-binding nucleotides.

Chronic stress enhanced fear memories are associated with increased amygdala zif268 mRNA expression and are resistant to reconsolidation.

The chronically stressed brain may present a vulnerability to develop maladaptive fear-related behaviors in response to a traumatic event. In rodents, chronic stress leads to amygdala hyperresponsivity and dendritic hypertrophy and produces a post traumatic stress disorder (PTSD)-like phenotype that includes exaggerated fear learning following Pavlovian fear conditioning and resistance to extinction. It is unknown whether chronic stress-induced enhanced fear memories are vulnerable to disruption via reconsolidation blockade, as a novel therapeutic approach for attenuating exaggerated fear memories. We used a chronic stress procedure in a rat model (wire mesh restraint for 6h/d/21d) to create a vulnerable brain that leads to a PTSD-like phenotype. We then examined freezing behavior during acquisition, reactivation and after post-reactivation rapamycin administration (i.p., 40mg/kg) in a Pavlovian fear conditioning paradigm to determine its effects on reconsolidation as well as the subsequent functional activation of limbic structures using zif268 mRNA. Chronic stress increased amygdala zif268 mRNA during fear memory retrieval at reactivation. Moreover, these enhanced fear memories were unaffected by post reactivation rapamycin to disrupt long-term fear memory. Also, post-reactivation long term memory processing was also associated with increased amygdala (LA and BA), and decreased hippocampal CA1 zif268 mRNA expression. These results suggest potential challenges for reconsolidation blockade as an effective approach in treating exaggerated fear memories, as in PTSD. Our findings also support chronic stress manipulations combined with fear conditioning as a useful preclinical approach to study a PTSD-like phenotype.

Solitary fibrous tumor - clinicopathologic, immunohistochemical and molecular analysis of 28 cases.

BACKGROUND: Solitary fibrous tumor is a mesenchymal tumor of fibroblastic type, which can affect any region of the body. Recently, a recurrent gene fusion NAB2-STAT6 has been identified as molecular hallmark. The NAB2-STAT6 fusion leads to EGR1 activation and transcriptional deregulation of EGR1-dependent target genes and is a driving event in initiation of SFT. In this study, we report the clinicopathologic and RT-PCR findings and evaluated expression of STAT6 and EGR1 protein in a cohort of 28 SFTs. METHODS: 28 patients with a median age of 54 years were included with SFTs originating at different sites, most occurring in the lung and pleura (9, 32%), 5 in soft tissues of the lower extremities (18%) and 5 in the head and neck (18%). For detection of the NAB2-STAT6 fusion gene, RT-PCR was performed using RNA extracted from formalin-fixed and paraffin-embedded tissues. Immunohistochemistry was performed on all cases with antibodies against STAT6 and EGR1. RESULTS: All patients were treated by surgery, 3 with adjuvant chemo- or radiotherapy. Follow-up data of 18 patients could be obtained of which 2 patients died of metastatic disease 13 months and 52 years after first diagnosis. Sixteen patients have no evidence of disease with a median follow up of 29.5 months (range 7 - 120 months). NAB2-STAT6 fusion transcripts were found in 19/28 cases (68%). The most common fusion was between NAB2 exon 4 and STAT6 exon 3 (11/19, 58%), mainly occurring in pleuropulmonary lesions. All cases showed strong nuclear expression of STAT6 (28/28, 100%) while EGR1 showed low-level variable nuclear expression in all samples, comparable with the EGR1 expression results of the control group. CONCLUSIONS: The identification of the NAB2-STAT6 fusion in SFTs can provide important diagnostic information, especially in cases with aberrant morphology or when biopsy material is limited. STAT6 immunohistochemistry is another useful tool in diagnosing SFT. EGR1 immunohistochemistry indicates low-level protein expression in accordance with EGR1 activation due to distorted NAB2 activity. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_224.

Extinction, applied after retrieval of auditory fear memory, selectively increases zinc-finger protein 268 and phosphorylated ribosomal protein S6 expression in prefrontal cortex and lateral amygdala.

Retrieval of consolidated memories induces a labile phase during which memory can be disrupted or updated through a reconsolidation process. A central component of behavioral updating during reconsolidation using a retrieval-extinction manipulation (Ret+Ext) is the synaptic removal of a calcium-permeable-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (CP-AMPARs) in the lateral amygdala-a metabotropic GluR1 receptor (mGluR1) dependent mechanism. In the present study, we investigate the effect of Ret+Ext on the expression of molecular markers that could play a role in the reconsolidation process. Specifically, we tested the effects of Ret+Ext on the global expression of zinc-finger 268 protein (Zif268), a marker previously found to be implicated in memory reconsolidation, to confirm its occurrence after retrieval (Ret) and Ret+Ext. We also evaluated the global expression of phosphorylated ribosomal protein S6 (rpS6P), here proposed as a marker of the mGluR1-mediated memory process induced by Ret+Ext. The expression of both markers (zif268, rpS6P) was assessed by immunolocalization in prelimbic cortex (PRL), infralimbic cortex (IL), ventral subdivision of the lateral amygdala (LA) and hippocampus CA1 (CA1) in fear-conditioned rats. Our results showed that retrieval and Ret+Ext, but not extinction alone, increased Zif268 expression in prefrontal cortex and lateral amygdala. Ret+Ext, but not retrieval, retrieval followed by context exposure or extinction alone, increased the expression of rpS6P in prefrontal cortex and LA. In summary, (i) Zif268 increased after retrieval confirming that reconsolidation is engaged in our conditions, (ii) Zif268 increased after Ret+Ext confirming that it does not simply reflect an extinction or reconsolidation disruption (Zif268 level of expression should be lower in both cases) and (iii) rpS6P increased after Ret+Ext, but not after extinction, suggesting, as expected, a potential mGluR1 mediated molecular mechanism specific for Ret+Ext. Together with the Zif268 increase, our results suggest that the Ret+Ext induced memory process is more similar to reconsolidation updating than extinction facilitation.

Egr-1 mediates chronic hypoxia-induced renal interstitial fibrosis via the PKC/ERK pathway.

BACKGROUND: Chronic hypoxia-induced epithelial-to-mesenchymal transition (EMT) is a crucial process in renal fibrogenesis. Egr-1, as a transcription factor, has been proven to be important in promoting EMT. However, whether it functions in hypoxia-induced renal tubular EMT has not been fully elucidated. METHODS: Egr-1 were detected at mRNA and protein levels by qPCR and Western blot analysis respectively after renal epithelial cells were subjected to hypoxia treatment. Meanwhile, EMT phenotype was also observed through identification of relevant EMT-specific markers. siRNA was used to knock down Egr-1 expression and subsequent changes were observed. Specific PKC and MAPK/ERK inhibitors were employed to determine the molecular signaling pathway involved in Egr-1-mediated EMT phenotype. In vivo assays using rat remnant kidney model were used to validate the in vitro results. Furthermore, Egr-1 expression was examined in the samples of CKD patients with the clinical relevance revealed. RESULTS: Hypoxia treatment enhanced the mRNA and protein levels of Egr-1 in HK-2 cells, which was accompanied by a reduced expression of the epithelial marker E-cadherin and an enhanced expression of the mesenchymal marker Fsp-1. Downregulation of Egr-1 with siRNA reversed hypoxia-induced EMT. Using the specific inhibitors to protein kinase C (calphostin C) or MAPK/ERK (PD98059), we identified that hypoxia induced Egr-1 expression through the PKC/ERK pathway. In addition, the upregulation of Egr-1 raised endogenous Snail levels, and the downregulation of Snail inhibited Egr-1-mediated EMT in HK-2 cells. Through in vivo assays using rat remnant kidney and CKD patients' kidney tissues, we found that Egr-1 and Snail were overexpressed in tubular epithelial cells with EMT. CONCLUSION: Egr-1 may be an important regulator of the development of renal tubular EMT induced by hypoxia through the PKC/ERK pathway and the activation of Snail. Targeting Egr-1 expression or activity might be a novel therapeutic strategy to control renal fibrosis.

Expression of early growth response 1 affects miR-106a/signal transducer and activator of transcription 3 regulating cognitive impairment in ovariectomized mice.

OBJECTIVE: This study aims to investigate the effects of early growth response 1 (Egr1) on miR-106a/signal transducer and activator of transcription 3 (STAT3) regulating cognitive impairment in an ovariectomy model. METHODS: Using the Morris water maze test, we assessed escape latency and time spent in a quadrant among mice at 6, 8, and 12 weeks after ovariectomy and their age-matched controls (n = 15 each group). Egr1, miR-106a, and STAT3 messenger RNA expression (n = 7) in the hippocampus and cortex of mice at 6, 8, and 12 weeks after ovariectomy was detected by quantitative real-time polymerase chain reaction, whereas Egr1, phospho-STAT3 (p-STAT3), and STAT3 protein expression (n = 8) was evaluated by Western blot analysis. Moreover, alterations in miR-106a and STAT3 expression were investigated in neuroblastoma (SH-SY5Y) cells transfected with a human Egr1 interference fragment (si-Egr1) or an Egr1-overexpressing plasmid (GV141-Egr1), respectively. RESULTS: Escape latency was significantly increased and time spent in a platform quadrant was reduced in mice at 12 weeks after ovariectomy compared with age-matched controls. Egr1 and miR-106a expression was obviously increased in the hippocampus and cortex at 12 weeks after ovariectomy, whereas STAT3 levels were decreased compared with 12-week controls. After SH-SY5Y cell transfection with the si-Egr1 fragment, miR-106a levels decreased and STAT3/p-STAT3 levels increased, whereas cotransfection of the miR-106a mimic caused a significant decrease in STAT3 levels. MiR-106a messenger RNA expression was significantly increased and STAT3/p-STAT3 protein levels were decreased by Egr1 overexpression, whereas simultaneous transfection with the miR-106a inhibitor inhibited alterations in STAT3 levels. CONCLUSIONS: This study suggests that Egr1 decreases STAT3 expression via miR-106a in ovariectomized mice with cognitive impairment, indicating that Egr1 represents a potential target for therapeutic intervention in postmenopausal cognitive decline.

Clinical significance of immunogenic cell death biomarker rage and early growth response 1 in human primary gastric adenocarcinoma.

The receptor for advanced glycation end products (RAGE), a pattern recognition receptor that binds multiple ligands derived from a damaged cell environment, contributes to multiple pathologies including cancer. Early growth response 1 (EGR1) is a tumor suppressor gene or a tumor promoter involved in tumorigenesis and progression of some cancers. However, there is some lack of knowledge about the expression and clinical significance of RAGE and EGR1 in human primary gastric adenocarcinoma (GAC). The present study was aimed to investigate the expression and clinical significance of RAGE and EGR1 in human GAC. One hundred and twenty cases of GAC tissues, adjacent non-cancer tissues (ANCT) and metastatic lymph node (MLN) tissues were collected. The expression of RAGE and EGR1 was assessed using immunohistochemistry (IHC) through tissue microarray procedure. The clinicopathologic characteristics of all patients were analyzed. As a result, the expression of RAGE in GAC and MLN tissues showed the positive staining mainly in the cytoplasm, with lower reactivity rate compared with the ANCT (P less than 0.001), while EGR1 expression had no significant difference between GAC, MLN tissues and ANCT (P=0.565). Moreover, the positive expression of RAGE was closely associated with the N stage of GAC patients, but did not correlate with their age, gender, tumor size, tumor sites, T stage, and metastatic lymph node (each P>0.05). In addition, Spearman Rank correlation analysis showed the positive correlation of RAGE expression with EGR1 in GAC tissues (r=0.658). Taken together, the expression of RAGE is decreased in GAC and MLN tissues, and is associated with the N stage of GAC patients, suggesting that RAGE may represent a potential therapeutic target for the treatment of GAC.

Hypochlorite-modified high-density lipoprotein promotes induction of HO-1 in endothelial cells via activation of p42/44 MAPK and zinc finger transcription factor Egr-1.

Modification/chlorination of high-density lipoprotein (HDL) by hypochlorous acid (HOCl), formed by the myeloperoxidase-H2O2-chloride system of activated phagocytes, converts an anti-atherogenic lipoprotein into a pro-inflammatory lipoprotein particle. Chlorinated HDL is present in human lesion material, binds to and is internalized by endothelial cells and impairs expression and activity of endothelial nitric oxide synthase. The present study aimed at clarifying whether exposure of endothelial cells to pro-inflammatory HOCl-HDL impacts on expression of heme oxygenase-1, a potential rescue pathway against endothelial dysfunction. Our findings revealed that HDL modified by HOCl, added as reagent or generated enzymatically, induced phosphorylation of p42/44 mitogen-activated protein kinase, expression of transcription factor early growth response-1 (Egr-1) and enhanced expression of heme oxygenase-1 in human endothelial cells. Upregulation of heme oxygenase-1 could be blocked by an inhibitor upstream of p42/44 mitogen-activated protein kinase and/or knockdown of Egr-1 by RNA-interference. Electrophoretic mobility shift assays demonstrated HOCl-HDL-mediated induction of the Egr-1 DNA binding activity. Immunocytochemical and immunoblotting experiments demonstrated HOCl-HDL-induced translocation of Egr-1 to the nucleus. The present study demonstrates a novel compensatory pathway against adverse effects of HOCl-HDL, providing cytoprotection in a number of pathological conditions including cardiovascular disease.

Suppression of osteosarcoma cell invasion by chemotherapy is mediated by urokinase plasminogen activator activity via up-regulation of EGR1.

BACKGROUND: The cellular and molecular mechanisms of tumour response following chemotherapy are largely unknown. We found that low dose anti-tumour agents up-regulate early growth response 1 (EGR1) expression. EGR1 is a member of the immediate-early gene group of transcription factors which modulate transcription of multiple genes involved in cell proliferation, differentiation, and development. It has been reported that EGR1 act as either tumour promoting factor or suppressor. We therefore examined the expression and function of EGR1 in osteosarcoma. METHODS: We investigated the expression of EGR1 in human osteosarcoma cell lines and biopsy specimens. We next examined the expression of EGR1 following anti-tumour agents treatment. To examine the function of EGR1 in osteosarcoma, we assessed the tumour growth and invasion in vitro and in vivo. RESULTS: Real-time PCR revealed that EGR1 was down-regulated both in osteosarcoma cell lines and osteosarcoma patients' biopsy specimens. In addition, EGR1 was up-regulated both in osteosarcoma patient' specimens and osteosarcoma cell lines following anti-tumour agent treatment. Although forced expression of EGR1 did not prevent osteosarcoma growth, forced expression of EGR1 prevented osteosarcoma cell invasion in vitro. In addition, forced expression of EGR1 promoted down-regulation of urokinase plasminogen activator, urokinase receptor, and urokinase plasminogen activity. Xenograft mice models showed that forced expression of EGR1 prevents osteosarcoma cell migration into blood vessels. CONCLUSIONS: These findings suggest that although chemotherapy could not prevent osteosarcoma growth in chemotherapy-resistant patients, it did prevent osteosarcoma cell invasion by down-regulation of urokinase plasminogen activity via up-regulation of EGR1 during chemotherapy periods.

Activation of the EP4 prostanoid receptor induces prostaglandin E2 and pro-inflammatory cytokine production in human airway epithelial cells.

Prostaglandin (PG)E2 mediates its effects via activation of four distinct PGE2 receptors, termed EP1₋4, all of which are present on the model human airway epithelial cell line, Calu-3. We previously reported that acute activation of the EP4 subtype of the PGE2 receptor is associated with increased anion efflux from these cells, via the CFTR chloride channel. In the present study we examine the effects of longer term activation of the EP4 receptor in Calu-3 cells in an attempt to determine whether this would prove beneficial or detrimental to the airway epithelial cell environment. Using PGE1-OH, an EP4 receptor selective agonist, we determined that EP4 receptor activation was associated with increased phosphorylation of extracellular signal-related kinases (ERKs) and induction of the transcription factor early growth response factor-1 (Egr-1). Additionally, using specific enzyme-linked immunosorbent assays and quantitative PCR, we detected increased production of PGE2, IL-6, IL-8 and the chemokine monocyte chemotactic protein-1 (MCP-1) at both the protein and gene level in response to EP4 receptor activation. Intriguingly, the enhanced production of PGE2 in response to EP4 receptor activation raises the possibility of a positive feedback situation. Generally, within the airways, PGE2 is considered to have pro-inflammatory effects, whilst the enhanced production of IL-6, IL-8 and MCP-1 would be associated with the recruitment and activation of inflammatory cells to the airways. Thus, we conclude that chronic activation of the EP4 receptor is associated with increased production of mediators likely to increase the pro-inflammatory milieu of airway epithelial cells.

Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells.

Early growth response-1 (Egr-1) is overexpressed in human prostate tumors and contributes to cancer progression. On the other hand, mutation of p53 is associated with advanced prostate cancer, as well as with metastasis and hormone independence. This study shows that in prostate cell lines in culture, Egr-1 overexpression correlated with an alteration of p53 activity because of the expression of SV40 large T-antigen or because of a mutation in the TP53 gene. In cells containing altered p53 activity, Egr-1 expression was abolished by pharmacological inhibition or RNAi silencing of p53. Although forced expression of wild-type p53 was not sufficient to trigger Egr-1 transcription, four different mutants of p53 were shown to induce Egr-1. Direct binding of p53 to the EGR1 promoter could not be detected. Instead, Egr-1 transcription was driven by the ERK1/2 pathway, as it was abrogated by specific inhibitors of MEK. Egr-1 increased the transcription of HB-EGF (epidermal growth factor), amphiregulin and epiregulin, resulting in autocrine activation of the EGF receptor (EGFR) and downstream MEK/ERK cascade. Thus, mutant p53 initiates a feedback loop that involves ERK1/2-mediated transactivation of Egr-1, which in turn increases the secretion of EGFR ligands and stimulates the EGFR signaling pathway. Finally, p53 may further regulate this feedback loop by altering the level of EGFR expression.

Early growth response-1 worsens ventilator-induced lung injury by up-regulating prostanoid synthesis.

RATIONALE: Ventilator-induced lung injury (VILI) is common and serious and may be mediated in part by prostanoids. We have demonstrated increased expression of the early growth response-1 (Egr1) gene by injurious ventilation, but whether-or how-such up-regulation contributes to injury is unknown. OBJECTIVES: We sought to define the role of Egr1 in the pathogenesis of VILI. METHODS: An in vivo murine model of VILI was used, and Egr1(+/+) (wild-type) and Egr1(-/-) mice were studied; the effects of prostaglandin E receptor subtype 1 (EP1) inhibition were assessed. MEASUREMENTS AND MAIN RESULTS: Injurious ventilation caused lung injury in wild-type mice, but less so in Egr1(-/-) mice. The injury was associated with expression of EGR1 protein, which was localized to type II cells and macrophages and was concentrated in nuclear extracts. There was a concomitant increase in expression of phosphorylated p44/p42 mitogen-activated protein kinases. The prostaglandin E synthase (mPGES-1) gene has multiple EGR1 binding sites on its promoter, and induction of mPGES-1 mRNA (as well as the prostanoid product, PGE2) by injurious ventilation was highly dependent on the presence of the Egr1 gene. PGE2 mediates many lung effects via EP1 receptors, and EP1 blockade (with ONO-8713) lessened lung injury. CONCLUSIONS: This is the first demonstration of a mechanism whereby expression of a novel gene (Egr1) can contribute to VILI via a prostanoid-mediated pathway.

Exploration of Emodin to treat alpha-naphthylisothiocyanate-induced cholestatic hepatitis via anti-inflammatory pathway.

Emodin, 1,3,8-trihydroxy-6-methyl-anthraquinone, is an anthraquinone derivative from the roots of Rheum officinale Baill that has been used to treat many diseases in digestive system for thousands of years. This study is to disclose the mechanism of Emodin to treat cholestatic hepatitis via anti-inflammatory pathway. Rats were divided into Emodin, ursodeoxycholic acid, Dexamethasone, model and blank control groups with treatment of respective agent after administration of alpha-naphthylisothiocyanate. At 24 h, 48 h and 72 h time points after administration, liver function, pathological changes of hepatic tissue, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), cytokine-induced neutrophil chemoattractant (CINC)-1, macrophage inflammatory protein (MIP)-2, intercellular adhesion molecule (ICAM)-1, nuclear factor (NF)-kappaB and early growth response (Egr)-1, nitric oxide (NO) and inducible nitric oxide synthase (iNOS) were detected. As a result, compared to the controls, Emodin had a notable effect on rat's living condition, pathological manifestation of hepatic tissue, total bilirubin, direct bilirubin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (P<0.05), but had little effect on alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT) and total bile acid. With Emodin intervention, levels of TNF-alpha, IL-6, MPO, MDA, CINC-1, MIP-2, ICAM-1 and translocation of NF-kappaB were remarkably decreased, and levels of NO and iNOS were markedly increased (P<0.05). Emodin had no effect on Egr-1. In conclusion, Emodin has a protective effect on hepatocytes and a restoring activity on cholestatic hepatitis by anti-inflammation. The effects are mainly due to antagonizing pro-inflammatory cytokines and mediators, inhibiting oxidative damage, improving hepatic microcirculation, reducing impairment signals, and controlling neutrophil infiltration.

Vascular endothelial sampling and analysis of gene transcripts: a new quantitative approach to monitor vascular inflammation.

BACKGROUND: Limited access to endothelial tissue is a major constraint when investigating the cellular mechanisms of vascular inflammation in patients with cardiovascular and metabolic diseases. We introduce venous endothelial sampling coupled to quantitative analysis of gene transcripts by real-time PCR, as a novel approach to study endothelial gene expression in human subjects. METHODS: Endothelial cells were collected from a superficial forearm vein using five guide wires sequentially inserted through a 20-gauge angiocatheter in seven patients with history of cardiovascular events related to advanced vascular disease and in 17 healthy subjects. Endothelial cells were purified using magnetic beads coated with endothelial specific antibodies. Endothelial mRNA was amplified using RiboAmp HS RNA Amplification kit (Molecular Devices, Sunnyvale, CA). Amplified RNA was analyzed by real-time PCR. RESULTS: Linearity of RNA amplification was validated by real-time PCR using RNA from 1,000 human umbilical endothelial cells (HUVECs) before and after amplification. In human subjects, vascular disease was associated with significant induction of proatherosclerotic genes: early growth response gene product (Egr-1) and monocyte chemoattractant protein-1 (MCP-1). CONCLUSION: Venous endothelial sampling coupled to real-time PCR analysis is a minimally invasive, safe, and reliable technique to monitor vascular inflammation in human subjects. Expression of genes implicated in the atherosclerotic process is increased in the venous endothelium of patients with arterial vascular disease. Venous endothelial sampling and quantitative analysis of gene expression may help develop new vascular-targeted biomarkers to identify and track the impact of disease states and therapeutic interventions in vascular diseases.

Egr-1-d2EGFP transgenic rats identify transient populations of neurons and glial cells during postnatal brain development.

The inducible transcription factor Egr-1 has been extensively studied in the adult brain but potential roles during development are largely unexplored. Here we describe the analysis of a new transgenic rat model (egr-1 promoter driving a destabilized GFP molecule) that has provided novel information about the postnatal roles of Egr-1. We show that Egr-1 is more widely expressed in the neonatal brain than was previously appreciated, and is not restricted to neurons; it is expressed in glial cells in the postnatal neocortex and hippocampus. This pattern of expression has been revealed due to cellular filling by GFP, permitting co-localization with glial markers. The transgene/Egr-1 is also expressed in a novel population of cells associated with Cajal-Retzius-like neurons within the marginal zone of the postnatal neocortex. Both of these cellular populations are transient, being limited to the neonatal period, before Egr-1 expression becomes established in an adult-like pattern within neocortical neurons, CA1 hippocampus, and striatum. Another transient population of transgene/Egr-1 cells in the bed nucleus of the stria terminalis is maintained until pre-adolescence. The transient phenotype of these cells involves a low relative expression of the neuronal marker NeuN, perhaps indicating a failure to achieve full neuronal differentiation. Egr-1 is therefore present in a diverse range of cell-types during postnatal development. Transgenic expression of a destabilized fluorescent marker has permitted identification of these novel cell populations and will facilitate further analysis of the transcriptional mechanisms that underlie the specific functions and fate of these cells during postnatal brain development.

Identification of gene networks modulated by activin in LbetaT2 cells using DNA microarray analysis.

Activins, members of the TGFbeta family of proteins, are widely expressed in a variety of tissues. First identified based on their ability to regulate biosynthesis and secretion of follicle-stimulating hormone (FSH), activins have also been shown to modulate development, cell growth, apoptosis, and inflammation. Despite their many known functions, the precise mechanisms and downstream signaling pathways by which activins mediate their diverse effects remain unknown. We have used a DNA microarray assay to identify genes that are regulated by activin, alone or in combination with gonadotropin-releasing hormone (GnRH), another major regulator of FSH, in a murine gonadotrope-derived cell line (LbetaT2). We used mRNA from these cells to screen Affymetrix Mu74av2 mouse Gene Chip oligonucleotide microarrays, representing approximately 12,400 mouse genes. Treatment of LbetaT2 cells with activin A, a gonadotropin-releasing hormone agonist (GnRHA) or activin A plus GnRHA resulted in alterations in levels of gene expression that ranged in magnitude from 15 to 67-fold. Data analysis identified 268 transcripts that were up- or down-regulated by two-fold or more. Distinct sets of genes were affected by treatment with activin, GnRHA and activin plus GnRHA, suggesting interactions between activin and GnRHA. Changes in expression of seven randomly selected representative genes identified by the microarray technique were confirmed by real-time quantitative PCR and semi-quantitative reverse transcription/PCR (RT/PCR). Modulation of expression of genes by activin suggests that activin may mediate its effects through a variety of signaling pathways.